Insulin-transferrin fusion protein and its prodrug, proinsulin-transferrin, for overcoming insulin resistance

ABSTRACT

A method of treating Type 2 diabetes (T2D) is provided. The method includes administering to a subject in need thereof an effective amount of a pharmaceutical composition that includes an insulin-transferrin fusion protein or its prodrug, proinsulin-transferrin fusion protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 USC § 371 National Stage application ofInternational Application No. PCT/US2018/014552 filed Jul. 13, 2018,which claims the benefit under 35 USC § 119(e) to U.S. Application Ser.No. 62/532,822 filed Jul. 14, 2017. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

FIELD OF THE INVENTION

The present invention relates generally to the treatment of metabolicdiseases and more specifically to the use of proinsulin-transferrin andinsulin-transferrin to treat diseases associated withinsulin-resistance, including type 2 diabetes.

BACKGROUND OF THE INVENTION

Type 2 diabetes (T2D) is a major health issue in the United States with9.3% of the population having this disease. World-wide, the prevalenceof diabetes is estimated to be about 8.5%. It is estimated that thetotal US population living with diabetes will increase 64% by 2025, anddiabetes-related Medicare expenditures will increase by 72% to $514billion/year. Moreover, diabetes is a major cause of blindness, kidneyfailure, heart attacks, stroke and lower limb amputation (Global reporton diabetes, World Health Organization, Geneva, 2016). Type 2 diabetesaccounts for 90-95% of all diagnosed cases of diabetes in adults.Therefore, there is an urgent need to find a treatment to either preventor cure T2D.

Studies on animal and clinical research have demonstrated that insulinresistance is the key mechanism leading to the development andpathogenesis of T2D, as well as many other diseases includingnon-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH) and Alzheimer's disease. It is generally agreed that overcominginsulin resistance is a major target for the prevention and treatment ofT2D. In insulin-resistant patients, cells become desensitized withinsulin and thus a higher level of insulin will be required to controlthe blood glucose level. The over demanding of insulin causes theexhaustion of insulin production by pancreatic β-islet cells andeventually leads to β-cell dysfunction.

Several medications, such as thiazolidinediones (TZDs), have beendeveloped as insulin-sensitizing drugs. However, the risk of adverseeffects with long-term use of these drugs is a safety concern. Severalof the TZDs, e.g., Actos, Avandia and Rezulin, have already beenwithdrawn from European and/or US market. Most insulin analogs currentlyin use in the treatment of T2D, such as Glargine and Detemer, only workon prolonging the plasma half-life of insulin, but not on increasinginsulin sensitivity. The persistence of insulin analogs in the bloodwill cause many insulin-side effects, including severe hypoglycemia andcardiovascular diseases.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a method of treatingType 2 diabetes (T2D) and other insulin resistance-associated diseasesincluding non-alcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH) and Alzheimer's disease. The method includesadministering to a subject in need thereof an effective amount of apharmaceutical composition that includes an insulin-transferrin fusionprotein (INS-Tf) or its prodrug, proinsulin-transferrin fusion protein(ProINS-Tf).

In one embodiment, the T2D, NAFLD, NASH and Alzheimer's diseases are allcaused by, or associated with, insulin resistance.

Another aspect of the present invention is directed to aligand-transferrin fusion protein. The ligand-transferrin fusion proteinis administered at an amount effective to cause an increase in theaffinity and duration of the ligand-receptor interaction on a cellsurface.

In one embodiment, the increase in the affinity and duration of theligand-receptor interaction on the cell surface results in an enhancedand prolonged biological activity of the ligand-receptor interaction.

In another embodiment, the invention provides a fusion protein encodedby a nucleic acid sequence comprising a pre-proinsulin nucleic acidsequence operably linked to a transferrin (Tf) sequence. The fusionprotein includes a pre-proinsulin protein that is cleaved to form aproinsulin-Tf protein (ProINS-Tf), which can be further converted to theactive form, insulin-transferrin (INS-TF). While not wanting to be boundby a particular theory it is believed that the bivalent binding to thetransferrin receptor and the insulin receptor may overcome insulinresistance.

In another embodiment, the invention provides a nucleic acid sequenceencoding a fusion protein described herein. In one aspect, the nucleicacid sequence is in a vector, such as a plasmid, a virus, a nanoparticleor a liposome. Similarly, the fusion protein may be in a deliveryvehicle for delivery to a subject in need thereof or a pharmaceuticallyacceptable carrier.

Insulin proteins and transferrin proteins as disclosed herein includevariants, homologs or analogs, for example, as long as such proteinshave a biological activity of a functional insulin protein or analogthereof and a functional transferrin protein.

The invention provides a method of treating a subject with type 2diabetes (T2D) comprising administering a fusion protein of theinvention (e.g., ProINS-Tf or INS-Tf) to a subject in need thereof in apharmaceutically acceptable carrier in an effective amount to reduceglucose levels in the subject, thereby producing ProINS-Tf or INS-Tf,respectively and treating the T2D. In one aspect, the subject was atleast partially resistant to insulin prior to treatment. In one aspect,the subject's blood glucose level is at a level of a non-diabeticsubject after treatment.

In another embodiment, the invention provides a method of producing aproinsulin-transferrin fusion protein in vitro comprising contacting ahost cell with a nucleic acid sequence encoding a fusion protein of theinvention and culturing the cell under conditions and for a time toproduce the fusion protein. For example, the fusion protein is furthertreated to produce insulin-transferrin. In one aspect, the treatment isby enzymatic digestion, for example, using trypsin or carboxypeptidase Bor a combination thereof. In another aspect, the treatment is bycultured hepatocyte or hepatocyte-like cells, including but not limitedto H4IIE cells. In one aspect, the host cell is a mammalian cell,including but not limited to a CHO cell or an HEK293 cell. In treatmentof a subject, the administration is in single-dosage form however, thiswill vary with chronic and acute indications. In one aspect, glucoselevels are reduced within about 4-8 hours of administration. In oneaspect, a second insulin drug, such as human insulin or analog thereof,for a human subject.

Other aspects and advantages of the invention will be apparent from thefollowing description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western Blot analysis of trypsin-digested ProINS-Tf withanti-Tf antibody and anti-INS B-chain antibody.

FIG. 2 shows the effect of INS and INS-Tf on the phosphorylation of Akt.The presence of a significant amount of p-Akt at 1 and 4 h after the 30min-pulse treatment at 4° C. with INS-Tf, but not with INS, indicated astronger and prolonged action of the fusion protein on IR.

FIGS. 3A and 3B show the induction of insulin-resistance in HepG2 cells.Insulin-resistance was induced by incubation of cells in serum-free DMEMwith different concentration of palmitate.

FIGS. 4A and 4B show overcoming of INS resistance by INS-Tf inpalmitate-induced INS resistant HepG2 cells.

FIGS. 5A-5C show graphs of blood glucose responses to insulin (INS)injection in NOD mice with normal glycemic and moderate or severehyperglycemic conditions.

FIG. 6 is a graph showing the response to ProINS-Tf treatment in insulinresistant NOD mice with severe hyperglycemia.

FIG. 7 illustrates the preproinsulin-Tf fusion gene construct in pcDNA3.1 (+) vector.

FIG. 8 shows the results of Western blot of ProINS-Tf fusion proteinusing anti-Tf and anti-proinsulin antibodies, demonstrating theexpression of both Tf and proinsulin in the fusion protein. Lane 1 andlane 2 are anti-Tf blot, and lane 3 is anti-proinsulin blot. Lane 1:apo-Tf. Lane 2 and lane 3: ProINS-Tf.

FIGS. 9A-9C show graphs for IR competitive binding profiles of INS andProINS-Tf

A) IR binding affinity of ProINS-Tf increased significantly after itsactivation. B) and C) Addition of excess Tf interrupted the binding ofINS-Tf to IR, suggesting that INS-Tf binding to IR was partiallyassisted by TfR binding.

FIG. 10A is a graph showing TfR-mediated conversion of ProINS-Tf toINS-Tf from various tissue slices TfR-mediated conversion was only seenin liver slices but not in other organs.

FIG. 10B is a graph showing the percentage of ¹²⁵I-ProINS-Tf associatedwith liver and muscle slices over time The amount of muscle slicesassociated ProINS-Tf decreased significantly after 1 h, while majorityof ProINS-Tf was still associated with liver slices after 1 h and 2 h.The prolonged retention of ProINS-Tf in liver slices but not muscleslices could be explained by the lack of conversion in muscle and thuslack of bivalent binding.

FIGS. 11A-11C are graphs showing the biodistribution of ¹²⁵I-labeledproteins in wild type CF-1 mice. The biodistribution of (A) Tf, (B)ProINS-Tf and (C) INS in major organs of CF-1 mice 1 h, 4 h and 8 h posti.v. injection. ProINS-Tf exhibited increased accumulation in liver overtime, suggesting its targeting effect to the liver, but not muscle. INShad relatively even distribution in liver and muscle, which are the twoINS-sensitive sites.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

As used herein, treating/treatment means any manner in which one or moreof the symptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein, such as use for treating a metabolicdisease.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular compound or pharmaceutical compositionrefers to any lessening, whether permanent or temporary, lasting ortransient that can be attributed to or associated with administration ofthe composition.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, cats, dogs and other domesticated animals as well asagriculturally important animals, e.g., bovine and porcine, and thelike, which is to be the recipient of a particular treatment. Preferablythe subject is a human.

As used herein, the term “protein precursor” refers to inactive proteinsor peptides that can be turned into an active form by posttranslationalmodification. An exemplary “protein precursor” may include proinsulin,proglucagon and proopiomelanocortin, but are not limited thereto.

As used herein, the term “prodrug” refers to a pharmacological substancethat is administered in an inactive or significantly less active form,but becomes activated in vivo through metabolic activities eitherintracellularly or extracellularly. Exemplary prodrugs may includeprohormones and other profactors.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “vector” refers to a nucleic acid construct designed fortransfer between different host cells. An “expression vector” refers toa vector that has the ability to incorporate and express heterologousDNA fragments in a foreign cell. Many prokaryotic and eukaryoticexpression vectors are commercially available. Selection of appropriateexpression vectors is within the knowledge of those having skill in theart. Accordingly, an “expression cassette” or “expression vector” is anucleic acid construct generated recombinantly or synthetically, with aseries of specified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

A “Tf domain” is a protein domain that retains the biological functionsof Tf, i.e., binding and transporting iron. In one embodiment, the Tfdomain may have the wild-type amino acid sequence of a Tf protein (e.g.,a human Tf protein). In other embodiments, the Tf domain may be avariant of the wild-type Tf. The activity of a Tf domain may bedetermined using any of the methods known in the art. For example, theactivity of a Tf domain may be determined by measuring its ability tobind a transferrin receptor (TfR).

As used herein “insulin” (INS) refers to native insulin, such as humaninsulin, insulin lispro, insulin aspart, regular insulin, insulinglargine, insulin zinc, human insulin zinc extended, isophane insulin,human buffered regular insulin, insulin glulisine, recombinant humanregular insulin, recombinant human insulin isophane, premixedcombinations of any of the aforementioned insulins, a derivativethereof, and a combination of any of the aforementioned insulins andanalogs thereof.

Formulation of Pharmaceutical Compositions

The pharmaceutical compositions provided herein contain therapeuticallyeffective amounts of one or more of compounds provided herein in apharmaceutically acceptable carrier.

The compositions contain one or more compounds provided herein. Thecompounds are preferably formulated into suitable pharmaceuticalpreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administration or in sterile solutions or suspensionsfor parenteral administration, as well as transdermal patch preparation,creams, ointments and dry powder inhalers. Typically the compoundsdescribed above are formulated into pharmaceutical compositions usingtechniques and procedures well known in the art (see, e.g., AnselIntroduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives is (are) mixed with asuitable pharmaceutical carrier or vehicle. The compounds may bederivatized as the corresponding salts, esters, enol ethers or esters,acids, bases, solvates, hydrates or prodrugs prior to formulation, asdescribed above. The concentrations of the compounds in the compositionsare effective for delivery of an amount, upon administration, thattreats, prevents, or ameliorates one or more of the symptoms ofconditions including, but not limited to, undesired cell proliferation,cardiovascular, renal, neurodegenerative/neurologic and ophthalmicdisorders, diseases or syndromes characterized by chronic inflammationand cardiovascular diseases as described herein.

Typically, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction ofcompound is dissolved, suspended, dispersed or otherwise mixed in aselected vehicle at an effective concentration such that the treatedcondition is relieved or ameliorated. Pharmaceutical carriers orvehicles suitable for administration of the compounds provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration.

In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients. Liposomal suspensions, includingtissue-targeted liposomes, such as tumor-targeted liposomes, may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known to those skilled in the art. For example,liposome formulations may be prepared as described in U.S. Pat. No.4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) maybe formed by drying down egg phosphatidyl choline and brain phosphatidylserine (7:3 molar ratio) on the inside of a flask. A solution of acompound provided herein in phosphate buffered saline lacking divalentcations (PBS) is added and the flask shaken until the lipid film isdispersed. The resulting vesicles are washed to remove unencapsulatedcompound, pelleted by centrifugation, and then resuspended in PBS.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in in vitro and in vivo systems described hereinand then extrapolated therefrom for dosages for humans.

The concentration of active compound in the pharmaceutical compositionwill depend on absorption, inactivation and excretion rates of theactive compound, the physicochemical characteristics of the compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to ameliorate one or more of the symptoms ofdiseases or disorders associated undesired cell proliferation,cardiovascular, renal, neurodegenerative/neurologic and ophthalmicdisorders, diseases or syndromes characterized by chronic inflammationand cardiovascular diseases as described herein.

Typically a therapeutically effective dosage should produce a serumconcentration of active ingredient of from about 0.1 ng/ml to about50-100 μg/ml. The pharmaceutical compositions typically should provide adosage of from about 0.001 mg to about 2000 mg of compound per kilogramof body weight per day. Pharmaceutical dosage unit forms are prepared toprovide from about 1 mg to about 1000 mg and preferably from about 10 toabout 500 mg of the essential active ingredient or a combination ofessential ingredients per dosage unit form.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

The amount of INS-Tf or ProINS-Tf administered will be dependent on thesubject being treated, the type and severity of the affliction, themanner of administration and the judgment of the prescribing physician.Although effective dosage ranges for specific biologically activesubstances of interest are dependent upon a variety of factors, and aregenerally known to one of ordinary skill in the art, some dosageguidelines can be generally defined.

Pharmaceutically acceptable derivatives include acids, bases, enolethers and esters, salts, esters, hydrates, solvates and prodrug forms.The derivative is selected such that its pharmacokinetic properties aresuperior to the corresponding neutral compound.

Thus, effective concentrations or amounts of one or more of thecompounds described herein or pharmaceutically acceptable derivativesthereof are mixed with a suitable pharmaceutical carrier or vehicle forsystemic, topical or local administration to form pharmaceuticalcompositions. Compounds are included in an amount effective forameliorating one or more symptoms of, or for treating or preventingdiseases or disorders associated with undesired cell proliferation,cardiovascular, renal, neurodegenerative/neurologic and ophthalmicdisorders, diseases or syndromes characterized by chronic inflammationand cardiovascular diseases as described herein. The concentration ofactive compound in the composition will depend on absorption,inactivation, excretion rates of the active compound, the dosageschedule, amount administered, particular formulation as well as otherfactors known to those of skill in the art.

The compositions are intended to be administered by a suitable route,including orally, parenterally, rectally, topically and locally. Fororal administration, capsules and tablets are presently preferred. Thecompositions are in liquid, semi-liquid or solid form and are formulatedin a manner suitable for each route of administration. Preferred modesof administration include parenteral and oral modes of administration.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution,polysorbate (TWEEN 80), fixed oil, polyethylene glycol, glycerine,propylene glycol or other synthetic solvent; antimicrobial agents, suchas benzyl alcohol and methyl parabens; antioxidants, such as ascorbicacid and sodium bisulfite; chelating agents, such asethylenediaminetetraacetic acid (EDTA); buffers, such as acetates,citrates and phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. Parenteral preparations can be enclosedin ampules, disposable syringes or single or multiple dose vials made ofglass, plastic or other suitable material.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as TWEEN®, or dissolution in aqueous sodium bicarbonate.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are typically formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms include ampulesand syringes and individually packaged tablets or capsules. Unit-doseforms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses, which are not segregated inpackaging.

The composition can contain along with the active ingredient: a diluentsuch as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acaciagelatin, glucose, molasses, polvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, or otherwise mixing an active compound as defined above andoptional pharmaceutical adjuvants in a carrier, such as, for example,water, saline, aqueous dextrose, glycerol, glycols, ethanol, and thelike, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, or solubilizing agents, pH buffering agents and thelike, for example, acetate, sodium citrate, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 15th Edition, 1975. The composition or formulationto be administered will, in any event, contain a quantity of the activecompound in an amount sufficient to alleviate the symptoms of thetreated subject.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier may beprepared. For oral administration, a pharmaceutically acceptablenon-toxic composition is formed by the incorporation of any of thenormally employed excipients, such as, for example pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, talcum, cellulosederivatives, sodium crosscarmellose, glucose, sucrose, magnesiumcarbonate or sodium saccharin. Such compositions include solutions,suspensions, tablets, capsules, powders and sustained releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid and others. Methodsfor preparation of these compositions are known to those skilled in theart. The contemplated compositions may contain 0.001%-100% activeingredient, preferably 0.1-85%, typically 75-95%.

The active compounds or pharmaceutically acceptable derivatives may beprepared with carriers that protect the compound against rapidelimination from the body, such as time release formulations orcoatings.

The compositions may include other active compounds to obtain desiredcombinations of properties. The compounds provided herein, orpharmaceutically acceptable derivatives thereof as described herein, mayalso be advantageously administered for therapeutic or prophylacticpurposes together with another pharmacological agent known in thegeneral art to be of value in treating one or more of the diseases ormedical conditions referred to hereinabove, such as diseases ordisorders associated with undesired cell proliferation, coronaryrestenosis, osteoporosis, syndromes characterized by chronicinflammation, autoimmune diseases and cardiovascular diseases. It is tobe understood that such combination therapy constitutes a further aspectof the compositions and methods of treatment provided herein.

Compositions for Oral Administration

Oral pharmaceutical dosage forms are either solid, gel or liquid. Thesolid dosage forms are tablets, capsules, granules, and bulk powders.Types of oral tablets include compressed, chewable lozenges and tabletswhich may be enteric-coated, sugar-coated or film-coated. Capsules maybe hard or soft gelatin capsules, while granules and powders may beprovided in non-effervescent or effervescent form with the combinationof other ingredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms,preferably capsules or tablets. The tablets, pills, capsules, trochesand the like can contain any of the following ingredients, or compoundsof a similar nature: a binder; a diluent; a disintegrating agent; alubricant; a glidant; a sweetening agent; and a flavoring agent.

Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, sucrose and starchpaste. Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

If oral administration is desired, the compound could be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activecompound in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar and otherenteric agents. The compounds can also be administered as a component ofan elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like.A syrup may contain, in addition to the active compounds, sucrose as asweetening agent and certain preservatives, dyes and colorings andflavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. The activeingredient is a compound or pharmaceutically acceptable derivativethereof as described herein. Higher concentrations, up to about 98% byweight of the active ingredient may be included.

Pharmaceutically acceptable carriers included in tablets are binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, and wetting agents. Enteric-coated tablets, because of theenteric-coating, resist the action of stomach acid and dissolve ordisintegrate in the neutral or alkaline intestines. Sugar-coated tabletsare compressed tablets to which different layers of pharmaceuticallyacceptable substances are applied. Film-coated tablets are compressedtablets which have been coated with a polymer or other suitable coating.Multiple compressed tablets are compressed tablets made by more than onecompression cycle utilizing the pharmaceutically acceptable substancespreviously mentioned. Coloring agents may also be used in the abovedosage forms. Flavoring and sweetening agents are used in compressedtablets, sugar-coated, multiple compressed and chewable tablets.Flavoring and sweetening agents are especially useful in the formationof chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicadd, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Diluents include lactose and sucrose. Sweetening agentsinclude sucrose, syrups, glycerin and artificial sweetening agents suchas saccharin. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelauryl ether. Organic additives include citric and tartaric acid.Sources of carbon dioxide include sodium bicarbonate and sodiumcarbonate. Coloring agents include any of the approved certified watersoluble FD and C dyes, and mixtures thereof. Flavoring agents includenatural flavors extracted from plants such fruits, and synthetic blendsof compounds which produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is preferablyencapsulated in a gelatin capsule. Such solutions, and the preparationand encapsulation thereof, are disclosed in U.S. Pat. Nos. 4,328,245;4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g.,for example, in a polyethylene glycol, may be diluted with a sufficientquantity of a pharmaceutically acceptable liquid carrier, e.g., water,to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. Re 28,819 and4,358,603. Briefly, such formulations include, but are not limited to,those containing a compound provided herein, a dialkylated mono- orpoly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Injectables, Solutions and Emulsions

Parenteral administration, generally characterized by injection, eithersubcutaneously, intrathecal, intrathecal, epidural, intramuscularly orintravenously is also contemplated herein. Injectables can be preparedin conventional forms, either as liquid solutions or suspensions; solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins. Implantation of aslow-release or sustained-release system, such that a constant level ofdosage is maintained (see, e.g., U.S. Pat. No. 3,710,795) is alsocontemplated herein. Briefly, a compound provided herein is dispersed ina solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The compound diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active compoundcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the compound and theneeds of the subject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN® 80). A sequestering or chelatingagent of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration.Typically a therapeutically effective dosage is formulated to contain aconcentration of at least about 0.1% w/w up to about 90% w/w or more,preferably more than 1% w/w of the active compound to the treatedtissue(s). The active ingredient may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the tissue being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the age of theindividual treated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of theformulations, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed formulations.

The compound may be suspended in micronized or other suitable form ormay be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

Lyophilized Powders

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving a compoundprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at,typically, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. Generally,the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage (10-1000 mg,preferably 100-500 mg) or multiple dosages of the compound. Thelyophilized powder can be stored under appropriate conditions, such asat about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, about 1-50 mg, preferably 5-35 mg, more preferably about9-30 mg of lyophilized powder, is added per mL of sterile water or othersuitable carrier. The precise amount depends upon the selected compound.Such amount can be empirically determined.

Topical Administration

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compounds or pharmaceutically acceptable derivatives thereof may beformulated as aerosols for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will typically have diameters ofless than 50 microns, preferably less than 10 microns.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

These solutions, particularly those intended for ophthalmic use, may beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts.

Compositions for Other Routes of Administration

Other routes of administration, such as topical application, transdermalpatches, and rectal administration are also contemplated herein.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The typical weight of a rectalsuppository is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

The present invention demonstrated the following:

Insulin-transferrin fusion protein can be produced from in vitroproteolytic digestion of proinsulin-transferrin fusion protein.

Insulin-transferrin fusion protein, but not proinsulin-transferrinfusion protein, exhibited a higher affinity and longer duration thaninsulin in insulin receptor binding on a cell surface.

Insulin-transferrin fusion protein can maintain an enhanced andprolonged insulin receptor response in insulin-resistant cells.

Insulin-transferrin fusion protein can overcome insulin-resistance ininsulin-desensitized cells.

Insulin-transferrin fusion protein can be used for the prevention ortreatment of type 2 diabetes.

Example 1 ProINS-Tf Recombinant Fusion Protein

ProINS-Tf recombinant fusion protein was produced as previouslydescribed in Wang Y, et al. J Control Release 2011; 155:386-392.Briefly, human preproinsulin sequence was ligated in frame withC-terminally his-tagged full-length human Tf. Plasmids containing thefusion gene were transfected to HEK 293 cells (ATCC, Manassas, Va.).Conditioned media were collected after 8-day cultures. Fusion proteinwas concentrated using tangential flow filtration (Millipore) andfurther purified by nickel nitrilotriacetic acid agarose (Qiagen,Valencia, Calif.). The fusion protein was characterized and quantifiedby SDS-PAGE followed by Coomassie blue staining and anti-Tf andanti-proinsulin Western blot. Recombinant human INS from Escherichiacoli (Sigma) was dissolved in 100 mmol/L HCl (pH 3.0) to 15 mg/mL andthen further diluted in PBS to a stock of 15 μg/mL. Recombinant humanproinsulin (ProINS; R&D Systems, Minneapolis, Minn.) was dissolved inPBS to a stock of 100 μg/mL. Recombinant INS glargine (Lantus) wasdiluted up to 13-fold in distilled water while maintaining the proper pHand zinc:INS ratio prior to in vivo use.

Example 2 Proinsulin-Tf Fusion Protein Expression and Characterization

Preproinsulin sequence (NM_000207) fused in frame with Tf sequence(NM_001063) was engineered into pcDNA3.1 (+) expression vector(Invitrogen, CA) by molecular cloning methods (FIG. 5). Plasmidscontaining preproinsulin-Tf fusion gene were transiently transfected toHEK 293 cells through polyethylenimine-mediated DNA transfection.Conditioned serum-free media were collected and concentrated by labscaletangential flow filtration system (Millipore, MA), and thenultrafiltered by Centricon (Millipore, MA). ProINS-Tf fusion protein wascharacterized and quantified by Western blot using both anti-Tf (Sigma,MO) and anti-(pro)insulin antibodies (Abcam, MA). Anti-Tf andanti-(pro)insulin Western blots demonstrated the presence of a majorband with molecular weight ˜89 kD, which indicated that ProINS-Tf fusionprotein was successfully expressed and secreted into media. Aleucine-glutamate dipeptide sequence was introduced between proinsulinand Tf due to the XhoI restriction enzyme cutting site. The Tf shown onLane 1 of FIG. 8 came from the original serum-free cell culture medium,CD 293 (Invitrogen), instead of production from transfected HEK293cells. The dipeptide linker remained stable during production process.

Example 3 INS-Tf Like Protein can be Generated Using Enzyme Digestion

ProINS-Tf (5 μg) was incubated with 0.5 μg of trypsin in 200 μL of PBS,pH 7, at 37° C. Trypsin reaction was stopped at different time points bythe addition of 1 μg Bowman-Birk inhibitor. Reaction products, after1:10 dilution to ˜30 nM, were analyzed by ProINS- or INS-specific RIAs,as well as Western Blot using anti-Tf and anti-INS B chain antibodies.As shown in Table 1, based on results from ProINS and INS-specific RIAs,the concentration of ProINS-Tf decreased and the concentration of INS-Tfincreased over the time of incubation, suggesting nearly all ProINS-Tfwas converted to INS-Tf like protein with 5 min of the trypsindigestion.

TABLE 1 ProINS-Tf/INS-Tf concentrations in trypsin-digested sample asdetermined by ProINS- and INS-specific Radioimmunoassay (RIA) Incubationtime (min) 0 1 3 5 ProINS-Tf (nM) 32.88 0.47 0.43 0.51 INS-Tf (nM) 10.1737.59 29.51 32.49

As disclosed in FIG. 1, Western blotting with anti-Tf indicated amolecular size shift from original ProINS-Tf to a slightly lowerposition, suggesting a small portion of the original protein had beenremoved. However, the quantity of Tf in the trypsin-digested sample didnot indicate any changes. In contrast, Western blotting with anti-INSB-chain antibody, which recognized INS B chain that is not present inProINS, showed a strong interaction with trypsin-digestion product butnot ProINS-Tf. Since anti-INS B-chain antibody recognized INS B-chain inINS that is not present in ProINS, this result indicated that an INS-Tflike product was produced.

Example 4 Effect of INS-Tf on the Activation of Insulin Receptor

Starved HepG2 cells were first incubated with serum-free DMEM containing1 nM INS or INS-Tf for 30 min at 4° C. (pulse phase), allowing proteinsto bind to the cell surface. After the pulse phase, cells were washedwith 4° C. PBS to remove any unbound proteins, then incubated in DMEMwithout INS or INS-Tf at 37° C. (chase phase). At indicated time points,cells were washed and lysed, and the cell lysates were subjected toWestern blotting analysis using anti phospho-Akt (p-Akt) antibodies.

In this pulse-chase experiment, all the Akt phosphorylation should bedue to the protein bound on the cell surface at 4° C. during the pulsephase and, therefore, can be used as an indirect measure of the relativebinding affinity and duration of either INS or INS-Tf to insulinreceptor. Results as shown in FIG. 2 indicated that INS-Tf exhibited aslightly higher activity than INS on Akt phosphorylation at 10 min afterthe incubation at 37° C. However, at 1 h and 4 h after incubation at 37°C., INS induced very little p-Akt while INS-Tf remained highly active.This result suggested that INS-Tf was bound to insulin receptor with asignificantly higher affinity and duration than INS.

Example 5 Induction of Insulin-Resistance in HepG2 Cells

HepG2 cells were starved in DMEM with or without various concentrationsof palmitate/BSA complex for 16 h. Starved HepG2 cells were thenstimulated by various concentrations of INS for 10 min. As shown in FIG.3(A), a decreased level of Akt phosphorylation indicated INS resistancethat had been developed in palmitate-treated HepG2 cells. Palmitateinduced INS resistance in a dose-dependent manner, withoutdown-regulating IR. FIG. 3(B) shows that, in INS-resistant HepG2 cells,INS can still induce a dose-dependent effect on Akt phosphorylation.However, a much higher concentration of INS is needed to induce asimilar level of Akt phosphorylation as compared with normal HepG2cells.

Example 6 Overcome Insulin-Resistance by INS-Tf in HepG2 Cells

As shown in FIG. 4(A), HepG2 cells were incubated with 0.25 mM ofpalmitate complex for 16 h, and the development of INS resistance wasconfirmed by decreased level of Akt phosphorylation when stimulatingwith the same treatment of INS or INS-Tf, i.e., 10 nM for 10 min. Levelsof p-Akt induced by INS or INS-Tf decreased by approximated 70% and 33%respectively in palmitate treated cells. Time-course Akt phosphorylationassay in INS resistant HepG2 cells is shown in FIG. 4(B). HepG2 cellswith INS resistance were treated with 1 nM of INS, 10 nM of INS, orINS-Tf converted from 10 nM of ProINS-Tf by incubation with H4IIE cellsas described previously (Wang Y, et. al. 2011 & 2014) for indicatedperiod of time. Phospho-Akt band density was normalized withcorresponding GAPDH band density. Data were presented as the averagevalues with error bars indicating the standard deviations (N=3).

Example 7 Develop Insulin-Resistance in Diabetic NOD Mice

Insulin was subcutaneously injected with the dose of 45 nmol/kg intothree groups of NOD mice (N=3 for each group) with severe diabetes (BGlevel above 500 mg/dL), moderate diabetes (BG level between 300-400mg/dL), and normal glycemia (BG level around 100 mg/dL). The bloodglucose level in each mouse was monitored at 1 h and 2 h post injection,with free feeding. As shown in FIG. 5, NOD mice with normal and moderateglycemic condition responded very well to 45 nmol/kg of insulintreatment at 1 h post injection, which lowered blood glucose level tonormal range (FIG. 5A), resulting in 55% and 80% of blood glucosereduction (FIG. 5B), respectively. However, NOD mice with severehyperglycemia did not respond to 45 nmol/kg of insulin treatment (FIGS.5A and 5B). The blood glucose concentration was not reduced 1 h postinjection, indicating NOD with severe hyperglycemia is resistant toinsulin treatment with the dose of 45 nmol/kg. Only a slight BG loweringeffect was observed in severe hyperglycemic NOD mice when the dose ofinsulin was increased from 45 to 180 nmol/kg (FIG. 5C), indicating ahigh insulin resistance was developed in this group of NOD mice.

Example 8 Overcome Insulin-Resistance by ProINS-Tf in Severe DiabeticNOD Mice

NOD mice with severe hyperglycemia as described in Example 7 wassubcutaneously injected with a single injection of ProINS-Tf (dose: 45nmol/kg, N=4), two injections of high dose of insulin (dose: 180nmol/kg, N=4) or two injections of PBS as control, and the blood glucoselevel was monitored during two 2 h feeding (grey area)/6 h fasting(clear area) cycles. As shown in FIG. 6, compared to the PBS or theinsulin treated group, the mice injected with a single dose of 45nmol/kg of ProINS-Tf exhibited much better blood glucose lowering effectunder fasting condition throughout the study. This result is comparableto that in non-resistant type 1 diabetic mice (Wang Y, Shao J, Zaro J L,and Shen W C; Diabetes. 2014 May; 63(5): 1779-1788), which indicatesthat ProINS-Tf is very effective in the control of basal glucose levelin insulin-resistant NOD mice.

Example 9 Binding Affinity

Protein preparation ProINS-Tf fusion protein was produced and purifiedas previously described. Activation of ProINS-Tf was conducted byincubating with H4IIE hepatoma cells and the active form of the fusionprotein, INS-Tf, was quantified using INS-specific radioimmunoassays(RIA).

Insulin Receptor (IR)-competitive binding affinity assays in HepG2 cellsReceptor grade tracers ¹²⁵I-Tyr(A14)-INS was incubated with variousconcentrations of unlabeled INS, ProINS-Tf or INS-Tf at 4° C. for 2 h.The amount of cell associated ¹²⁵I-Tyr(A14)-INS was measured afterintensive washing. 100-fold excess Tf was included to investigate theinterference caused by the loss of TfR binding. (FIGS. 9A, 9B, and 9C).

FIGS. 9A-9C show graphs for IR competitive binding profiles of INS andProINS-Tf. (A): IR binding affinity of ProINS-Tf increased significantlyafter its activation. (B) and (C): Addition of excess Tf interrupted thebinding of INS-Tf to IR, suggesting that INS-Tf binding to IR waspartially assisted by TfR binding.

TABLE 2 IC₅₀ values extrapolated from the IR competitive binding curvesProINS-Tf had the lowest affinity to IR among the three proteins,indicating its low activity on triggering IR pathways. The bindingaffinity of INS-Tf to IR was 8.5-fold higher than that of INS, but theaffinity decreased in the presence of excess Tf. INS ProINS-Tf INS-TfIC₅₀(nM), no Tf 1.8 566 0.213 IC₅₀(nM), 100xTf 1.19 — 14.7

Example 10 Biodistribution of ProINS-Tf, Tf and INS

Ex vivo conversion and retention in precision-cut tissue slices. Organsamples including liver, intestine, lungs, kidneys, muscle and braincollected from wild type CF-1 mice were precisely sliced at thethickness of 250 μm. Freshly prepared tissue slices were treated with 10nM ProINS-Tf and the conversion product INS-Tf was detected fromaliquots of incubation medium using INS specific RIA. To measure theretention of ProINS-Tf in liver and muscle slices, tissue slices werefirst incubated in 10 nM ¹²⁵I-ProINS-Tf solution for 4 h, then chased inmedium containing 10 μM Tf. Tissue slices associated fusion protein wasmeasured by a gamma counter.

FIG. 10A is a graph showing TfR-mediated conversion of ProINS-Tf toINS-Tf from various tissue slices TfR-mediated conversion was only seenin liver slices but not in other organs. FIG. 10B is a graph showing thepercentage of ¹²⁵I-ProINS-Tf associated with liver and muscle slicesover time The amount of muscle slices associated ProINS-Tf decreasedsignificantly after 1 h, while majority of ProINS-Tf was stillassociated with liver slices after 1 and 2 h. The prolonged retention ofProINS-Tf in liver slices but not muscle slices could be explained bythe lack of conversion in muscle and thus lack of bivalent binding.

FIGS. 11A-11C are graphs showing the biodistribution of ¹²⁵I-labeledproteins in wild type CF-1 mice. The biodistribution of (A) Tf, (B)ProINS-Tf and (C) INS in major organs of CF-1 mice 1, 4, and 8 h posti.v. injection. ProINS-Tf exhibited increased accumulation in liver overtime, suggesting its targeting effect to the liver, but not muscle. INShad relatively even distribution in liver and muscle, which are the twoINS-sensitive sites. Biodistribution study CF-1 mice were administeredwith 5 nmole/kg of ¹²⁵I-INS, ¹²⁵I-Tf or ¹²⁵I-ProINS-Tf through tail vaininjection. Mice were sacrificed at 1, 4 or 8 h post injection and majororgans were collected. The amount of organ-associated radioactiveproteins was counted and normalized to whole blood.

Although the present invention has been described in terms of specificexemplary embodiments and examples, it will be appreciated that theembodiments disclosed herein are for illustrative purposes only andvarious modifications and alterations might be made by those skilled inthe art without departing from the spirit and scope of the invention asset forth in the following claims

REFERENCES

The following references are each relied upon and incorporated herein intheir entirety.

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What is claimed is:
 1. A method of treating insulin resistance in asubject with diabetes having insulin resistance comprising administeringa fusion protein of proinsulin and transferrin (ProINS-Tf) or insulinand transferrin (INS-Tf) to a subject in need thereof in apharmaceutically acceptable carrier in an effective amount to reduceblood glucose levels in the subject, thereby treating insulin resistancein the subject.
 2. The method of claim 1, wherein the subject was atleast partially resistant to insulin prior to treatment.
 3. The methodof claim 1, wherein the subject's blood glucose level is at a level of anon-diabetic subject after treatment.
 4. The method of claim 3, whereinglucose levels are reduced within about 4-8 hours of administration. 5.The method of claim 1, wherein administration is in single-dosage form.6. The method of claim 1, further comprising administering a secondinsulin drug.
 7. The method of claim 1, wherein the subject is a human.8. A method of treating a subject with insulin resistance comprisingadministering a fusion protein encoded by a nucleic acid sequencecomprising a pre-proinsulin nucleic acid sequence operably linked to atransferrin (Tf) sequence to a subject in need thereof in apharmaceutically acceptable carrier thereby treating the insulinresistance.
 9. The method of claim 1 or 8, wherein the subject has Type2 Diabetes (T2D).